Helix 8 Leu in the CB1 cannabinoid receptor contributes to selective signal transduction mechanisms
Helix 8 Leu in the CB1 cannabinoid receptor contributes to selective signal transduction mechanisms
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DOI：
10.1074/jbc.m703388200
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发表时间：
2007-08-24
影响因子：
4.8
通讯作者：
Abood, Mary E.
中科院分区：
文献类型：
--
作者：
Anavi-Goffer, Sharon;Fleischer, Daniel;Abood, Mary E.
CB1大麻素受体的胞内C末端螺旋8 （H8）偏离高度保守的NPXXY(X)(5,6) F G-蛋白偶联受体基序，具有Leu而不是Phe。我们比较了CB1与L7.60F突变和L7.60I突变的信号转导能力。在[S-35]鸟苷5 ' -3 - O-（硫代）三磷酸结合试验中，两种突变受体调节G-蛋白的能力与野生型（WT）不同。L7.60F受体在激动剂WIN- 55,212- 2和CP- 55,940的刺激下表现出减弱，而HU- 210则没有，而L7.60I受体在所有激动剂以及反向激动剂利莫那班的刺激下表现出减弱。突变体的内化速度比WT受体快，但同样可以从生长抑素受体中隔离G-蛋白。在突变体中，WIN- 55,212- 2对N型Ca2（+）电流的抑制时间和最大抑制量均有所降低。在L7.60I突变体中，对百日破毒素不敏感的G-蛋白进行重组实验，发现与G α (i3)偶联的缺失，而与G α (0A)偶联的缺失，在L7.60F突变体中，时间过程的减少是由G α (i3)控制的。此外，G α (i3)而不是G α (0A)增加了基础促进比，表明G α (i3)负责CB1的强直活性。共免疫沉淀研究显示两种突变受体均与G α (i1)或G α (i2)相关，但与G α (i3)无关。对WT CB1受体和各突变体1-棕榈酰- 2-油基磷脂酰胆碱双分子层的分子动力学模拟表明，每种突变体的H8包装是不同的。沿着每个H8的螺旋骨架的氢键模式也不同，H8的肘部区域的几何形状也不同(R7.56（400)- K7.58(402)）。本研究表明，NPXXY(X) (5,6) L的进化修饰有助于CB1受体的最大活性，并为与化学上不同的激动剂观察到的差异偶联提供了分子基础。
The intracellular C- terminal helix 8 ( H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY( X)(5,6) F G- protein- coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type ( WT) in their ability to regulate G- proteins in the [ S-35] guanosine 5 ' - 3- O-( thio) triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN- 55,212- 2 and CP- 55,940 but not HU- 210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G- proteins from the somatostatin receptor. Both the time course and maximal N- type Ca2(+) current inhibition by WIN- 55,212- 2 were reduced in the mutants. Reconstitution experiments with pertussis toxin- insensitive G- proteins revealed loss of coupling to G alpha (i3) but not G alpha (0A) in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by G alpha (i3) .Furthermore, G alpha (i3) but not G alpha(0A) enhanced basal facilitation ratio, suggesting that G alpha(i3) is responsible for CB1 tonic activity. Co- immunoprecipitation studies revealed that both mutant receptors were associated with G alpha(i1) or G alpha(i2) but not with G alpha(i3). Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1- palmitoyl- 2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 ( R7.56( 400)- K7.58( 402)). This study demonstrates that the evolutionary modification to NPXXY( X) (5,6) L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.